Diagnosis of Mycobacterium bovis infection

ABSTRACT

A method for the diagnosis of Mycobacterium bovis infection in a susceptible animal, comprises detection in said animal of antibodies against the MPB-70 protein of M.bovis and/or the detection of a cell-mediated immune response of said animal to the said MPB-70 protein. Also disclosed is a recombinant DNA molecule corresponding to all or portion of the M.bovis DNA sequence coding for the MPB-70 protein or a polypeptide having the antigenicity of MPB-70 protein, or degenerate forms thereof, as well as recombinant MPB-70 protein or polypeptide and a process for the preparation thereof.

This application is a Continuation of Ser. No. 08/104,927, filed Aug.12, 1993; which is a Continuation of Ser. No. 07/585,094, filed asPCT/AU89/00143 Mar. 31, 1989 (abandoned).

This invention relates to the diagnosis of bovine tuberculosis, and inparticular it relates to the use of a particular species-specificantigen for the diagnosis of Mycobacterium bovis infection, such asbovine tuberculosis, by both antibody and cellular assays.

Bovine tuberculosis (BTB) is a major disease of cattle worldwide. In theAmericas alone it was estimated to have cost the cattle industry $83million in 1977 (World Health Organisation, 1983). Several nations havemounted or are running campaigns to eliminate BTB, and although thesecampaigns have drastically reduced the incidence of the disease, nonehave been totally successful. In many parts of the world there is noconcerted effort to control the disease, which poses human, as well asanimal, health risks. The causative agent of bovine tuberculosis,Mycobacterium bovis, is closely related to M.tuberculosis.

Eradication of the disease from cattle has been hampered by the lack ofsensitivity and specificity of the bovine skin test currently used fordetection of infected animals. A simple serological test for BTB wouldbe the preferred option, but there are two fundamental problems with anytest for M.bovis-specific antibody. Infected animals generally have lowlevels of antibody to M.bovis, and many of the antibodies producedcross-react with antigens from other environmental mycobacterial ornocardial species (Daniel et.al., 1978). Enzyme-linked immunoassaysusing M.bovis protein extracts as antigens have been used to detectinfected cattle (Ritacco et.al., 1987; Theon et.al., 1983), but thesetests using crude antigens appear to lack sufficient specificity orsensitivity to be acceptable for use in an eradication campaign (Auer,1987). These problems are common to the serological diagnosis of allmycobacterial infections.

The MPB-70 protein is a major antigen of Mycobacterium bovis andM.bovis/BCG strains and can constitute more than 10% of the totalprotein present in culture filtrate preparations from these organisms.(Nagai et.al., 1981; Harboe and Nagai, 1984). It has been shown thatthis protein induces a potent delayed skin test reaction in M.bovis/BCGimmunized guinea pigs and has limited cross-reactivity with otherspecies of Mycobacteria, (Harboe et.al., 1986; Haslov, et.al., 1987).

In work leading to the present invention, the utility of this antigen inantibody and cellular assays for the diagnosis of M.bovis infections incattle has been examined. The MPB-70 protein was found to be expressedby all bovine field isolates of M.bovis tested (151 separate fieldisolates, Table 1) and was not present in various other mycobacteriacommonly found in the environment.

Accordingly, the present invention provides a method for the diagnosisof Mycobacterium bovis infection in susceptible animal, which ischaracterised by the detection in said animal of antibodies against theMPB-70 protein of M.bovis and/or the detection of a cell-mediated immuneresponse of said animal to the said MPB-70 protein.

In essence, the present invention is based on the use of the MPB-70protein as a specific antigen for the diagnosis of M.bovis infection.MPB-70 may be used as an antigen in a test for antibody to M.bovis inaccordance with any of the commonly known antibody tests, such as ELISA,RIA, CFT methods, and the like. MPB-70 may also be used as an antigen inthe caudal fold skin test in vivo, as well as an antigen in in vitroassays for cell-mediated immune responses such as proliferation assaysor assays based on the release of gamma interferon or interleukin 2.

Whilst the present invention is particularly directed to the diagnosisof M.bovis infection in cattle, causing bovine tuberculosis, the methodof the invention extends to detection of M.bovis infection in any othersusceptible animal species, including for example, deer, badgers,possums, pigs and camels.

The present invention also extends to a process for the purification ofMPB-70 from M.bovis culture filtrate material, as well as to theproduction of recombinant MPB-70 by cloning and expression in a hostcell such as E.coli.

It will of course be appreciated that the references to MPB-70 proteinherein include not only the full protein, but also any polypeptidesderived therefrom which have the antigenicity of MPB-70 and thereforecan be used in the assays described herein in the same manner as MPB-70protein itself.

The MPB-70 protein has been purified from an M.bovis culture filtrate bychromatofocusing on a Mono-P column. FIG. 1 shows a typical elutionprofile with the arrowed peaks containing the majority of the MPB-70antigen. When the fractions in each peak were pooled and probed onWestern blots with a monoclonal antibody specific for MPB-70, a proteinof 22 kd molecular weight was detected (FIG. 2).

Purified MPB-70 has been examined using both in vivo and in vitrocellular assays. The in vivo tests were carried out using theconventional skin test in both guinea pigs sensitized with killedM.bovis cells and cattle infected with live M. bovis. MPB-70 induced apositive reaction in both immunised guinea pigs (Table 2) and infectedcattle (Table 3) although not as strong as that induced by bovinetuberculin Purified Protein Derivative (PPD) at similar concentrations.MPB-70 has also been tested in vitro for the ability to stimulateproliferation of peripheral blood lymphocytes from M.bovis infectedcattle. The level of reactivity to MPB-70 was similar in all the M.bovisinfected animals and there was no activity with lymphocytes fromuninfected control cattle (Table 4).

In order to ascertain its suitability as a specific antigen fordetection of M.bovis infected cattle, MPB-70 has been tested in anantibody assay (ELISA). Crude culture filtrate antigen was used forcomparison in the antibody assay and the antibody results were comparedwith the conventional in vivo caudal fold skin test. Although thesensitivity of the MPB-70 ELISA was less than that achieved with boththe crude antigen ELISA and the caudal fold test, the specificity ofthis assay was far superior with only a few false positive results(Table 5). The low sensitivity of the antibody assay was most likely dueto the fact that mycobacteria preferentially induce a T cell rather thana humoral response (Thorns and Morris, 1983).

In further investigations, the MPB-70 gene from M.bovis has been clonedinto the λgtll expression vector and its DNA sequence determined. Thesequence of MPB-70 so determined differs considerably from that deducedby Patarroyo et.al., (1986; 1986a) using protein sequencing methods(FIG. 3). When serum samples from M.bovis infected cattle were screenedby Western blotting on a variety of recombinant M.bovis fusion proteins,MPB-70 was shown to be the dominant antigen recognised (Table 6). Therecombinant MPB-70 protein has also been found to induce good cellularresponses in M.bovis immunized guinea pigs and cattle.

Determination of the DNA sequence of the MPB-70 gene enables productionof DNA probes corresponding to all or a portion of this DNA sequence,and the use of such a probe for the detection of M.bovis or M.bovis/BCGorganisms in samples such as cultures, sputum or tissue samples.Accordingly, in another aspect of this invention there is provided amethod for the detection of M.bovis or M.bovis/BCG organisms in asample, which comprises the steps of contacting said sample with a DNAprobe corresponding to all or a portion of the DNA sequence of theMPB-70 gene, and detecting binding of said probe to indicate thepresence of said organisms in said sample.

                  TABLE 1                                                         ______________________________________                                        Immunoperoxidase Staining or Mycobacterium bovis and other bacteria           with a monoclonal antibody specific for MPB-70                                Positive         Negative                                                     ______________________________________                                        M. bovis strain TMC.sup.1 410                                                                  M. africanum strain TMC 5122                                 M. bovis strain AN5                                                                            M. microti strain TMC 601                                    M. bovis strain PMC.sup.2 203                                                                  BCG.sup.3 strain Glaxo                                       M. bovis strain PMC 205                                                                        BCG strain Copenhagen                                        M. bovis strain PMC 259                                                                        M. tuberculosis strain H37Rv                                 M. bovis field strains                                                        CSIRO 50         (CSIRO daughter strain)                                      S.A..sup.5 78    M.A.I.S..sup.4 serovars 2,6,8,9,10,15,18                     Vic..sup.6 16    M. gordonae                                                  Qld..sup.7 2     M. phlei                                                     N.Z..sup.8 5     M. kansasii                                                  BCG strain Moreau                                                                              M. paratuberculosis                                          BCG strain Tokyo M. flavescens                                                BCG strain Pasteur                                                                             Nocardia asteroides                                          BCG strain CSL.sup.9                                                                            Brucella melitensis strain 16M                              M. tuberculosis strain H37Rv                                                                   B. abortus strain 19                                         (Qld daughter strain)                                                                          Rhodoccccus equi                                             M. tuberculosis strain DT                                                                      R. rhodochrous                                               M. tuberculosis strain C                                                      M. tuberculosis strain PN                                                     M. tuberculosis field strains - 7                                             ______________________________________                                         .sup.1 TMC = Trudeau Mycobacteria Collection                                  .sup.2 PMC = Parkville Mycobacteria Collection                                .sup.3 BCG = Bacille CalmetteGuerin                                           .sup.4 M.A.I.S. = Mycobacterium aviumintracellularescrofulaceum complex       .sup.5 S.A. = South Australia                                                 .sup.6 Vic = Victoria                                                         .sup.7 Qld = Queensland                                                       .sup.8 N.Z. = New Zealand                                                     .sup.9 CSL = Commonwealth Serum Laboratories                             

                  TABLE 2                                                         ______________________________________                                        Delayed skin response to bovine PPD.sup.(1) and to purified MPB-70            in guinea pigs sensitized with killed M. bovis strain AN5.sup.(3).                           Response                                                                Concentration                                                                             Diameter mm Area mm.sup.2                                Antigen  ng/50 μl (2)                                                                           (mean + S.D.)                                                                             (mean + S.D.)                                ______________________________________                                        Bovine PPD                                                                             350.0       12.62 ± 1.51                                                                           169.0 ± 32.3                                       35.0        9.80 ± 1.47                                                                            98.5 ± 29.6                                        3.5         2.16 ± 3.49                                                                            14.8 ± 26.1                               MPB-70   350.0       12.2 ± 1.10                                                                            149.8 ± 27.7                                       35.0         7.9 ± 0.89                                                                            65.4 ± 16.8                                        3.5         0.0         0.0                                          ______________________________________                                         .sup.(1) concentration of PPD is based on known biological activity.          .sup.(2) concentration of purified antigens is based on freezedried           weight.                                                                       .sup.(3) control animals did not show any response.                      

                  TABLE 3                                                         ______________________________________                                        Delayed skin response to bovine PPD and purified MPB-70 in cattle             infected with live M. bovis.                                                           PPD          MPB-70                                                                           Skin           Skin                                  Animal                   thickness      thickness                             No.   M. bovis+                                                                              Diam (mm) (mm.sup.2)                                                                           Diam (mm)                                                                             (mm.sup.2)                            ______________________________________                                        213   I.V.     40        14.7   35      9.6                                   221   I.T.     65        20.6   45      9.6                                   229   I.T.     40        7.1    11      16.0                                  250   I.V.     40        14.1   35      9.2                                   253   I.V.     35        10.0   25      6.8                                   259   I.T.     70        14.5   40      10.1                                  230   Nil      0         0.1    0       0.1                                   265   Nil      0         4.3    0       1.2                                   ______________________________________                                         Bovine PPD 0.1 mg,                                                            MPB70 0.08 mg,                                                                +10.sup.6 M. bovis injected either intravenously (I.V.) or intratrachealy     (IT)                                                                     

                  TABLE 4                                                         ______________________________________                                        Proliferative response or bovine lymphocytes from M. bovis infected           cattle to bovine PPD and purified MPB 70.                                                       Antigen                                                     Animal    Immunization  PPD        MPB-70                                     ______________________________________                                         81       AN5 immunized 11.8        9.1                                       219       NIL            1.0        1.0                                       230       NIL            1.5(1.1).sup.#                                                                           0.7(1.1)                                  220       i.t. .sup.+ M. bovis                                                                        16.1(23.0)  5.7(8.7)                                  252       i.t. M. bovis 10.2(37.3)  2.9(5.2)                                  275       i.t. M. bovis 28.8(60.4)  4.7(9.5)                                  269       i.v. .sup.+ M. bovis                                                                        60.6(87.6) 13.5(17.6)                                 277       i.v. M. bovis 43.4(41.3)  5.4(5.6)                                  ______________________________________                                         M. bovis PPD 20 μg/ml.                                                     purified MPB70 50 μml.                                                     Animals tested 8 weeks post infection.                                        .sup.# Results reported as stimulation indexes from two separate              experiments.                                                                  .sup.+ i.t. = intratracheal, i.v. = intravenous.                         

                  TABLE 5                                                         ______________________________________                                        Comparison of an antibody assay using MPB-70 or crude antigen with            the caudal fold test.                                                                     Caudal fold                                                                             MPB-70     Crude antigen                                Test Results                                                                              Test      ELISA      ELISA                                        ______________________________________                                        True Positive                                                                             85        40         68   culture                                                                       positive                                False Negative                                                                            11        56         28   animals                                 True Negative                                                                             49        92         59   culture                                                                       negative                                False Positive                                                                            50        7          40   animals                                 ______________________________________                                    

The Infected status of all cattle was determined by extensive culture oflymph node and lesioned material.

                  TABLE 6                                                         ______________________________________                                        Western blot analysis of M. bovis fusion proteins with sera                   from M. bovis infected cattle.                                                Number of sera                                                                             Molecular weight of cloned antigen                               tested       19k    22k        65k  70k                                       ______________________________________                                        47           1.sup.#                                                                              36         4    3                                         ______________________________________                                         .sup.# number of sera giving a positive signal                                MPB70 = 22k protein.                                                     

The 19, 65 and 70k proteins rove previously been cloned and described(Shinnick et al, 1987)

Further features of the present invention will be apparent from thefollowing detailed Examples and the accompanying drawings.

In the drawings:

FIG. 1 shows separation of M.bovis antigens on a Mono-P column bychromatofocusing. Freeze dried culture filtrate, approx. 15 mg wasdissolved in 0.5 ml of 1:10 Polybuffer PB-74, pH 4.0 and applied ontothe column. Fractions were analysed by SDS-PAGE and Western blotting formonoclonal antibody reactivity. Arrowed peaks contain reactiveantigen(s). The fractions for each of these peaks were combined.

FIG. 2 shows SDS-PAGE and Western blotting analysis of pooled antigensfrom the Mono-P column. 30 μl of each pool was loaded.

(a) gel stained with silver stain.

(b) Western transfer of similar gel probed with M.bovis specificmonoclonal antibody (SB10).

Lanes (1) mol wt markers, (2) culture filtrate, (3) pool a, (4) pool b,(5) pool c and (6) pool d.

FIG. 3 shows the sequence of the M.bovis AN5 of MPB-70 gene, and itstranslated protein sequence.

FIG. 4 shows probing of various mycobacterial DNA with a MPB-70 geneprobe.

EXAMPLE 1 Isolation and Purification of MPB-70 antigen from M.bovis AN5cultures.

1. Mycobacterial cultures were grown in BAI medium for 12 weeks in 5%CO₂ in air at 37° C.

2. The culture medium was collected and centrifuged to remove cells anddebris. It was then filtered successively through 0.45 μm and twicethrough 0.22 μm Millipore membranes.

3. The culture filtrate (CF) was concentrated tenfold by ultrafiltration(Amicon YM-5) or by reverse osmosis against Aquacide II (Calbiochem).The buffer was exchanged for dist. H₂ O and the antigens werelyophilized.

4. The lyophilized material was dissolved in a small volume of a tenfolddilution of Polybuffer PB-74 (Pharmacia) which was adjusted to pH 4.0with imino-diacetic acid (IMDAA). Precipitated antigens were removed bycentrifugation and the supernatant was applied onto a chromatofocusingcolumn (Pharmacia Mono-P, 0.5×20 cm, or a 1.5×60 cm column packed withPharmacia PBE-94 gel) which was equilibrated with 0.025M piperazineadjusted to pH 5.6 with IMDAA. The antigens were eluted with ≈14 columnvolumes of PB-74 diluted tenfold and adjusted to pH 4.0 with IMDAA. Theelution was monitored by absorbance at 280 nm.

5. Fractions containing MPB-70 were pooled and further purified by gelfiltration. When minor contaminants were present the preparation wasfurther purified by passing it through a Sepharose Con A column and/or aSuperose 12 (Pharmacia) column.

EXAMPLE 2 Use of Purified MPB-70 antigen in Assays

(a) MATERIALS AND METHODS

Antisera and Monoclonal Antibodies. Sera from immunized animals, fromnaturally or experimentally M.bovis infected animals, and fromnon-infected control animals were used.

Cattle were immunized by repeated subcutaneous injection of 10 mg ofkilled M.bovis (AN5 strain) in 1 ml of saline. Cattle were also infectedwith live M.bovis (AN5) by either intravenous (i.v.) or intratracheal(i.t.) injection of 10⁶ bacteria.

Monoclonal antibodies against irradiated M.bovis sonicate, were raisedand kindly provided by Agen Biomedical Ltd. They have been characterizedand designated SB1-SB10 (Wood et.al., 1988). ELISA. Polystyrenemicrotitre trays (Nunc Immuno Plate) were coated with 100 μl of 3 μg/mlof M.bovis culture filtrate or, 10 μg/ml of purified antigen inphosphate buffered saline (PBS) (pH 7.2) overnight at 4° C. Plates werethen washed 4 times in PBS with Tween 20 (PBST), blocked with 0.1%sodium casein, and sera diluted in PSBT were added. Trays were incubatedfor 90 min at room temperature washed and reincubated with an antibovineIgG antibody conjugated to horseradish peroxidase (HRP:Miles).

After 90 min the trays were washed and incubated with substrate(2,2'-azino-di(3-ethylbenzthiazoline sulphonate)! (ABTS) for 30 min.Results were read at 405 nm in a Titertek Multiskan.

Immunization acid skin testing of guinea pigs. Guinea pigs weresensitized with a suspension of killed and dried M.bovis strain AN5 in aparaffin oil at a concentration of 0.5 mg/ml. An initial dose of 0.4 mlwas given by injecting 0.1 ml subcutaneously at each of four sites. Abooster injection of 0.2 ml was given intradermally at two sites 37 dayslater. Skin testing was conducted 40 days after the second inoculation.

For each antigen two groups of five guinea pigs were used. One groupconsisted of sensitized animals and the other non-sensitized controls.Each guinea pig was inoculated intradermally with 50 μl of each of threetenfold dilutions of only one antigen. The response to this antigen wasassessed at 48 hours by measuring the diameter and area of erythema.

The responses to the purified antigens were compared to an equal weightof bovine tuberculin PPD (Commonwealth Serum Laboratories, Aust.).

Preparation of peripheral blood lymphocytes (PBL). Ten to 20 ml of bloodwas collected into vacutainers (Becton-Dickinson) containing heparin (20units/ml) or sodium citrate (3.8%). The blood was then centrifuged at800 g for 20 min, the bully coat removed, diluted up to 10 ml with Hanks(GIBCO:Ca⁺⁺, Mg⁺⁺ free) and overlayed onto 10 ml of lymphopaque (BDH:1.086 g/ml). After centrifuging at 800 g for 25 min the interphase celllayer was collected and washed twice (450×g; 10 min) with 20 ml Hanks.The cells were finally resuspended in 5 ml RPMI 1640 (GIBCO) and viablecounts done using eosin (0.2%) exclusion.

Lymphocyte Proliferation Assay. Isolated lymphocytes were cultured inflat-bottom 96 well trays (Nunc) at 2.5×10⁵ cells/well in RPMIcontaining 5% foetal calf serum, L-glutamine, 2-Mercaptoethanol andantibiotics. After 48 hour incubation with antigen (25 μl/well) thecultures were pulsed with tritiated thymidine (Amersham; 0.5 μCi/well)and harvested 24 hours later using an automatic cell harvester(Skatron). The amount of tritiated thymidine incorporated was determinedusing an appropriate scintillant by counting in a liquid β scintillationcounter. Results were expressed as mean counts per minute (CPM) oftriplicate cultures, and the stimulation index calculated as shownbelow. ##EQU1## SDS-Polyacrylamide Gel Electrophoresis. Antigens werecharacterised by their electrophoretic mobility on 15% polyacrylamidegel cast on a Bio-Rad Protean II apparatus. Three centimeters of 4%polyacrylamide stacking gel was used. The buffer system was that ofLaemmli (1970). Electrophoresis was carried out at room temperature at20 mA per gel through the stacking gel and then at 25 mA per gel throughthe separating gel. In some experiments a Bio-Rad mini gel apparatus wasused. The buffers and gel concentrations were as above. Electrophoresiswas carried out at 150 volts for approx. 1.5 hours. The gels werestained with Coomassie Brilliant Blue (CBB) and/or with silver stain(Bio-Rad).

Immunoblotttng. Antigens from SDS-PAGE, gels were transferredelectrophoretically onto nitrocellulose membranes according to themethod of Towbin et. al. (1979). The membranes were probed for two hourswith antisera or monoclonal antibodies diluted in PBST. They were thenincubated with HRP-conjugated sheep anti-bovine, or anti-mouse IgG(Silenus), followed by the HRP substrate (4-chloro-1-naphthol) until thereactive bands were visible.

(b) RESULTS

Purification of single components containing the specific determinantswas achieved by chromatofocusing on a Mono-P column as described inExample 1. FIG. 1 shows a typical elution profile of this step. Thearrowed peaks contain most of the antigen that binds to the M.bovisspecific monoclonal antibodies but all the peaks eluted after the firstarrowed peak (a) contain material that reacts with the monoclonalantibodies. The mol.wt. of the reactive antigen in peak (a) isapprox.20K, while the antigens in all the following peaks were 22K. Thefractions in each arrowed peak were pooled. FIG. 2(a) shows the SDS-PAGEpatterns of these pools and FIG. 2(b) shows the reaction of the antigenswith the monoclonal antibody (SB10), after they were transferred ontonitrocellulose. Western transfers of the purified antigens probed withserum from an M.bovis infected animal, also show single reactive bands(result not shown).

The elution pattern varied somewhat for different CF batches showingconsiderable variation in their content of the antigen in relation tothe total protein (FIG. 2(a)). The amounts of the higher mol.wt. speciespresent, which contained the reactive epitopes, also varied. In somecases they were only minimal (FIG. 2(a)) and in others quiteconsiderable. The lower the relative amounts of these higher species,the better the recovery of the 22K protein. When the higher species wererelatively abundant, small amounts could be recovered in thechromatofocusing purification, often co-purifying with the 22K bands.They were removed if the CF was first passed through a Con A-Sepharosecolumn. This suggests that the larger molecules contain a sugar moiety.The Con A Sepharose step improved the recovery of the 22K protein.

Gel filtration was used to remove additional contaminants whennecessary.

Antigens from pools a, b and c were tested in an ELISA system for theirsuitability for diagnostic use. Crude CF was used as a comparison. Apanel of sera of M.bovis infected and non-infected animals were testedas well as some sera from animals infected with related mycobacteria andother bacteria.

Sera from M.bovis infected animals that gave high ELISA values with theCF were tested against the purified antigens. In this experiment onlyantigens `b` and `c` were examined, however in earlier experiments itwas found that antigen `a` gave similar ELISA readings to `b` (resultnot shown).

MPB-70 was also examined in CMI tests both in vivo and in vitro. The invivo tests were carried out using the conventional skin test on guineapigs which were sensitized with M.bovis cells. MPB-70 gave a positivereactions although it was not as active as PPD at the lowerconcentrations tested (Table 2).

MPB-70 was also tested in vitro for its ability to induce proliferationof peripheral blood lymphocytes from M.bovis infected cattle. Theprotein induced similar levels of reactivity in all the M.bovis infectedanimals and showed no activity with lymphocytes from uninfected controlanimals. The absolute level of reactivity of MPB-70 was less than thatseen with M.bovis PPD antigen and varied considerably between individualanimals.

EXAMPLE 3 Cloning of M.bovis AN5 DNA into λgt ll and Detection of ClonesExpressing Specific Antigens of M.bovis

(a) MATERIALS AND METHODS

Phage and bacteria. λgtll, Escherichia coli Y1089, and E.coli Y1090 havebeen previously described (Young & Davis, 1983), as has the pEXexpression vector (Stanley & Luzio, 1984). M.bovis AN5 was obtained fromthe Commonwealth Serum Laboratories, Parkville, Australia. TheCommonwealth Serum Laboratories obtained the strain from the Ministry ofAgriculture Weybridge Laboratories, England, in 1973. Subsequently, ithas been stored in freeze-dried ampoules.

Enzymes. All enzymes were purchased from Promega Biotec. Except wherespecifically mentioned, all of the DNA techniques used were as describedby Maniatis et.al., (1982).

MAbs. MAbs to M.bovis were a generous gift from Agen BiomedicalAustralia Ltd. The properties of MAbs SB1 to SB10 are publishedelsewhere (Wood et.al., 1988).

Isolation of M.bovis DNA. M.bovis AN5 was grown on BAI medium (Patersonet.al., 1958) for 6 weeks, after which cells were harvested bycentrifugation. DNA extraction was essentially by the method ofShoemaker et.al. (1986).

Construction of the M.bovis library in λgtll. M.bovis DNA was sonicatedbriefly (˜3secs) at low power, giving DNA fragments ranging in size from2 kilobase pairs to 200 base pairs (bp), as assessed by agarose gelelectrophoresis. DNA methylation, flushing with T4 DNA polymerase, andaddition of EcoRI linkers were by the protocol of Young et.al. (1985).Elimination of excess EcoRI linkers after linker ligation and EcoRIdigestion was achieved by using gel filtration (Superose 12 column;Pharmacia), and the eluate was monitored by UV absorption. The DNA wasthen ethanol precipitated and suspended in the TE buffer, and 0.5 μg wasligated with 1 μg of dephosphorylated EcoRI-digested λgtll (Promega)overnight at 4° C. Phage packaging was in Stratagene gigapack extracts.

Preparation of affinity-purified antibody to M.bovis. An emulsion ofapproximately 2×10⁹ heat-killed mycobacterial cells in Freund incompleteadjuvant was used for hyperimmunization of rabbits. Rabbits received twoinoculations, 35 days apart. Blood was taken from the ear of each rabbit10 days after the second inoculation. Sera were collected, and the titerto M.bovis PPD (Commonwealth Serum Laboratories) was determined byenzyme-linked immunosorbent assay. Rabbit sera used in affinitypurification gave titers of 1:40,960, as determined by enzyme-linkedimmunosorbent assay. An M.bovis affinity column was prepared by couplingM.bovis PPD to Sepharose 4B by the method of March et.al, (1974). Thecolumn (20 ml) was equilibrated with 3 volumes of affinity buffer (0.1MTris, 0.5M NaCl, 0.1% Tween 20), 5 ml of rabbit serum was pumpedthrough, and the column was then washed with 3 volumes of affinitybuffer to elute unbound material. Bound substances were then eluted with2M glycine hydrochloride (pH 2.5) into 2M Tris, and the eluate wasmonitored by UV absorption. Eluted protein was concentrated by using anAmicon concentrator and washed with phosphate-buffered saline (PBS), andthe protein concentration was determined by the method of Bradford(1976).

Screening of the λgtll library. Filters were taken from 85 mm-diameterphage overlay plates prepared as described by Young et.al., (1985).Immediately after removal from the plates, filters were washed for 10min. in PBST and then blocked in PBS-5% bovine lacto transfer techniqueoptimizer (skim milk) for 1 h. Affinity-purified antibody to M.bovis wasadded to 3.5 μg ml⁻¹, and the filters were shaken gently overnight to 4°C. The filters were then sequentially washed for 10 min in PBS, PBST,and PBS, after which swine anti-rabbit horseradish peroxidase conjugatewas added tin PBST (Daiko; 1/300) and the filters were incubated withgentle rocking for 2 h. Washing was repeated, and 4-chloro-1-naphtholsubstrate was added. Colour development was complete after 15 min. Thefilters were then washed in distilled water and dried.

Plaques corresponding to reactive spots on the filters were picked offand placed in 1.0 ml of SM medium. Purification was by spotting of 5-μlsamples from serial dilutions of the original phage suspensions on lawnsof E.coli Y1090 for further antibody probing, after which singlereactive plaques were picked, suspended and streaked on lawns of E.coliY1090.

When Mabs were used for analysis of isolated clones, preparation offilters was identical to the method described. MAbs were substituted foraffinity-purified serum (1/2,000 ascites), and anti-mouse alkalinephosphatase conjugate (Promega; 1/5,000) was used for detection. Filterswere developed in 5-bromo-4-chloro-3-indolyl-phosphate-Nitro BlueTetrazolium substrate.

Protein electrophoresis and Western blotting (Immunoblotting). Proteinsamples were solubilized by heating at 100° C. for 5 min in Trishydrochloride (pH 6.8) containing 2% sodium dodecyl sulfate, 5%(vol/vol) glycerol, and 0.002% (wt/vol) bromophenol blue. Vertical slabsodium dodecyl sulfate-polyacrylamide gel electrophoresis was carriedout essentially as described by Laemmli (1970), in 12% acrylamide gels.Proteins were transferred to nitrocellulose by electro-blottingovernight. Probing with MAbs (1/2,000) was done by the protocol used forphage spots. Probing with cattle sera was done at 1/50 dilution in PBSTfor 2 h, and the sera were absorbed with pEX-derived protein bound tonitrocellulose for 1 h before use. Bovine antibodies were detected byusing a peroxidase-conjugated antibovine immunoglobulin MAb (AustralianMonoclonal Developments).

Sequencing. Sequence analysis was done by the primer extension dideoxytermination method of Sanger et.al. (1980) after subcloning in M13tg130(Amersham Corp.); the products of the sequencing reaction were analysedon 6% polyacrylamide-8M urea gels.

(b) RESULTS

Construction of a λgtll library of M.bovis. M.bovis AN5 DNA wasextracted, sonicated, and cloned into λgtll. Australian PPD for use inthe bovine caudal-fold-skin test is isolated from M.bovis AN5, and sincePPD is a proven diagnostic reagent, this was the strain chosen forextraction of DNA for cloning. The AN5 library contained 2.5×10⁶independent PFU, of which 90% were recombinant as assessed byβ-galactosidase inactivation. The sizes of the inserts, as measured byrestriction digestion and agarose gel analysis of the library, rangedfrom 200 to 900 bp.

Probing the M.bovis AN5 library with polyclonal sera. Twenty filtersrepresenting 5×10⁴ PFU per filter (10⁶ PFU overall) were probed withaffinity-purified polyclonal anti-M.bovis AN5 serum. Between two andeight reactive phage plaques were found on each filter, and these werepicked off the plates and further purified. The signal strength of eachplaque varied greatly, ranging from barely perceptible dots toconspicuous spots. Not all of the filter spots revealed furtherreactions on phage purification; of the 88 spots found on the originalfilters, 32 phage gave consistent signals and passed through primary andsecondary purification steps. Very little background due to E.colicross-reactive antibody was found by using the affinity-purifiedantibodies, in contrast to the situation when unpurified sera fromeither rabbits or cattle were used. Absorption with E.coli lysate eitherbound to nitrocellulose or added directly to serum was far lesseffective in reducing background signal than the use ofaffinity-purified antiserum.

Analysis of recombinant clones with MAbs. Purified phage preparationsand lysogens of the λgtll clones reactive with polyclonal antibody wereprobed with M.bovis-M.tuberculosis-specific MAbs SB1 to SB10 in phagespot and colony assays to check for the presence of the specificepitopes.

Of the 32 recombinant phage, 5 expressed antigens that were recognisedby at least one of the MAbs when phage spots were probed, although asmight be expected of antigens derived from a randomly cloned library,not all of these five clones were recognised by all of the MAbs (Table7). Since MAbs SB1 to SB10 are known to recognise three separateepitopes on the protein antigen MPB70 (Wood et.al., 1988), this isindicative that the recombinant antigens recognised by the MAbs arecoded by clones derived from separate ligation events carrying insertsrepresenting different sections of the MPB70 gene.

Of the five λgtll clones recognised by the MAbs, two designated pB3C andC4a, displayed affinity for all of the MAbs in the panel, with theexception of MAb SB9, which did not recognize clone pB3C. Agarose gelanalysis showed that clone C4a had an EcoRI fragment insert of ˜250 bp.This was the smallest inserted fragment found that carried all of thespecific epitopes and was capable of coding for less than half of MPB70(molecular weight, 22,000). As such it was possible that this clonecarried none of the cross-reactive epitopes thought to be present onMPB70 (Hathoe & Nagai, 1984) and might prove useful as an antigen inserological diagnosis of BTB.

                  TABLE 7                                                         ______________________________________                                        Recognition of M. bovis clones by SB MAbs*                                    Reactivity with the following clones:                                         MAb    pB2a       pB3c   C4a      XB2a XC2a                                   ______________________________________                                        SB1    -          +      +        -    -                                      SB2    -          +      +        +    -                                      SB3    -          +      +        -    -                                      SB4    -          +      +        -    -                                      SB5    -          ±   +        -    -                                      SB6    -          +      +        +    -                                      SB7    -          +      +        +    +                                      SB8    -          ±   +        -    -                                      SB9    -          -      +        -    -                                      SB10   +          +      +        +    +                                      ______________________________________                                         *Reactivity was assessed by probing of phage spots. Drops (5 μl)           containing -10.sup.3 phage were spotted on E. coli Y1090 lawns, and           filters were prepared and probed as described in the text. The symbol .+-     indicates a weak reaction.                                               

To facilitate serologlcal analysis, the C4a insert was cloned into thehigh-expression pEX vector (Shoemaker et.al., 1986), and expression wasconfirm by immunoblotting of colonies with the SB10 NAb. The pEX vectorsexpress cloned proteins as a cro-β-gal fusion protein, insoluble in mostaqueous solutions, which allows for a relatively simple initialpurification, eliminating most soluble proteins by washing in 1% NonidetP-40-1% deoxycholate in PBS. After the washed pEX C4a fusion protein wasdissolved in sodium dodecyl sulfate loading buffer, the mixture was runon an acrylamide gel before transfer to nitrocellulose. Sera from bothM.bovis-infected and healthy cattle were used to probe the Westernblots. These blots indicated that antibody reactive to the fusionprotein was limited to infected animals. However, not all animals withthe disease had a detectable antibody response to the cloned protein.Neither pooled sera from BTB-free herds nor six sera from uninfectedcattle in a BTB-infected herd showed reactions with the cloned protein,although it was necessary to absorb out anti-E.coli activity in the seraand to use a specific monoclonal antibovine immunoglobulin conjugate tolower cross-reactivity.

Sequence of the MPB-70 gene. Synthetic peptides containing the specificepitopes of MPB70 have diagnostic potential and should be totally freeof endogenous cross-reactions. To obtain the DNA sequence of the MPB-70gene, and consequently the peptide sequence of the MPB-70, a clone ofthe MPB-70 gene was isolated. The insert from λgtll clone C4a was usedto probe a long-fragment library of M bovis AN5 constructed in thevector EMBL3. After purification of an EMBL3 clone reactive by DNAhybridization with the λgtll C4a insert, DNA was extracted from theEMBL3 clone and analysed by restriction enzyme digestion and agarose gelelectrophoresis. Southern blot analysis, again using the λgtll insert asa probe, showed the MPB-70 gene to be located on a 1.85 Kbpr Pstlrestriction fragment. Subcloning of the 1.85 Kbpr Pstl fragment into thebacteriophage M13 permitted the elucidation of the sequence of theMPB-70 gene. FIG. 3 shows the DNA sequence and inferred protein sequenceof the mature protein MPB-70. This protein is produced intracellularlywith a signal sequence that is cleared from the mature molecule.

Patarroyo et.al., (1986, 1986a) published two quite different peptidesequences for MPB-70 which were obtained by protein sequencingtechniques. The sequence described in FIG. 3 aligns with Pattaroyo's(1986a) up to amino acid 112, with 13 variations. From amino acids 112to 163 there is no similarity. A similar situation occurs with thesequence of MPB-70 given in Pattaroyo (1986). In this case thesimilarity with our DNA translated sequence (FIG. 3) ends at amino acid78, prior to which there are seven variations. Following on from aminoacid 80 and discounting a 17 amino acid sequence of Pattaroyo's (1986)which is not present in the DNA translated sequence (FIG. 3) thesesequences coincide up to amino acid 130, with eight variations. Fromamino acid 130 to 163 they are totally different.

EXAMPLE 4

Diagnosis of M.bovis infections in deer

Sera from M.bovis infected deer were tested in an ELISA using the MPB-70protein as antigen. A pool of sera from non-infected deer was used as anegative control and serum from a known M.bovis infected cow was used asa positive control. A commercially available monoclonal antibody tobovine IgG, conjugated to horseradish peroxidase (Bi2, AustralianMonoclonal Development, Sydney) was found to also bind cervine antibodyand was therefore used as a conjugate in this system.

In this MPB-70 ELISA all six infected deer had antibody levelssignificantly above that of the control serum (Table 8). Thirty-twoindividual sera from tuberculosis free deer were also tested in thissystem and found to be negative. (a) Method

The ELISA system used to test cervine sera was the same as that used fortesting bovines, however two steps in the procedure were altered: (1)the blocking step prior to antibody binding was omitted and (2) amonoclonal anti-bovine IgG conjugate was used instead of a polyclonalconjugate.

                  TABLE 8                                                         ______________________________________                                        MPB-70 ELISA with M. bovis-infected Deer Sera                                 Infected Sera  Optical Density                                                ______________________________________                                        1              2.9                                                            2              2.9                                                            3              0.9                                                            4              0.5                                                            5              2.8                                                            6              0.5                                                            Negative control                                                                             0.1                                                            Positive control                                                                             2.9                                                            ______________________________________                                    

All sera from infected deer were obtained from New Zealand. M.bovisinfection was confirmed by histology and bacteriological culture oftissues from the animals.

EXAMPLE 5 Use of M.bovis DNA sequence as hybridisation probe

(a) Method

The mycobacteria listed were cultured in 50 ml of Dubos broth media andharvested by centrifugation. Cell pellets were heat-killed at 70° C. andstored frozen at -20° C. until required. Nucleic acid was extracted fromthe cells as follows. Each of the cell pellets were resuspended in TEbuffer to a total volume of 1 ml. The cell suspensions were sonicatedfor 5 seconds on ice. Proteinase K was added to give a finalconcentration of 60 μg/ml and the suspensions were incubated at 65° C.for 1 hour. The samples were extracted once with 1 volume of phenolsaturated with TE, then once with Leder phenol (50% phenol, 50% CIAchloroform:isoamyl alcohol, 24:1!), once with an equal volume of CIA andfinally with 1 volume of water saturated ether. The nucleic acid wasethanol precipitated and resuspended in 25 μl of water. Concentration ofDNA and RNA in the extracts were measured spectroscopically. Aliquots ofapproximately 200 ng were applied to Hybond-N membrane using a dot blotapparatus. The DNA was denatured and fixed to Hybond-N as described bythe manufacturer. The blot was prehybridised and hybridised in astandard solution, including Plotto and sheared herring sperm DNA. Theprobe used was the 1.85 kb Pstl fragment that contains the gene forMPB-70, labelled with ³² P using a standard method of oligonucleotidepriming. Hybridisation was carried out at 37° C. over night. The blotwas washed in 1.0 SSC, 0.1% SDS at 65° C. prior to autoradiography.

(b) Results

Various DNA preparations of the mycobacteria listed below were probed indot blots with a radioactive DNA probe of the MPB-70 gene. As can beseen from FIG. 4, all and only M.bovis isolates showed binding of theprobe indicating the presence of an homologous MPB-70 gene andsurrounding region:

Row (a):

1. M.bovis AN5

2. M.bovis AN5

3. Field isolate a, Nth.Terr. M.bovis

4. Field isolate b, Nth.Terr. M.bovis

5. Feral pig isolate, M.bovis

6. Victorian abattoir isolate, M.bovis

7. New Zealand possum isolate, M.bovis

8. M.bovis BCG

Row (b)

1. MAIS 2

2. MAIS 2

3. M.phlei

4. M.phlei

5. M.kansasii

6. M.kansasii

7. MAIS 8

8. MAIS 8

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We claim:
 1. A method for the detection of Mycobacterium bovis infectionin a susceptible animal, which comprises detecting in said animal animmune response to MPB-70 protein of M.bovis having the amino acidsequence of FIG. 3 by means of an assay selected from the groupconsisting of an assay for MPB-70 recognizing antibodies and an assayfor a cell-mediated immune response; said assay using said MPB-70protein or an antigenic polypeptide derived therefrom reactive withaffinity-purified antibody to M. bovis as antigen, and said MPB-70protein or antigenic polypeptide derived therefrom being substantiallyfree of other M.bovis proteins.
 2. A method according to claim 1,wherein said animal is selected from the group consisting of cattle,deer, badgers, possums, pigs and camels.
 3. A method according to claim1, wherein a cell-mediated immune response of said animal is detected byan in vivo caudal fold skin test using said MPB-70 protein or anantigenic polypeptide derived therefrom as an antigen.
 4. A methodaccording to claim 1, wherein a cell-mediated immune response of saidanimal is detected by an in vivo assay using said MPB-70 protein orantigenic polypeptide derived therefrom as antigen.
 5. A methodaccording to claim 4, wherein said in vitro assay is a lymphocyteproliferation assay or an assay based on release of gamma interferon orinterleukin².
 6. A kit for the detection of Mycobacterium bovisinfection in a susceptible animal, which comprises means for thedetection in said animals of an immune response to MPB-70 protein ofM.bovis having the amino acid sequence of FIG. 3 by means of an assayselected from the group consisting of an assay for MPB-70 recognizingantibodies and an assay for cell mediated immune response, said kitcomprising said MPB-70 protein or an antigenic polypeptide derivedtherefrom reactive with affinity-purified antibody to M.bovis asantigen, and said MPB-70 protein or antigenic polypeptide derivedtherefrom being substantially free of other M.bovis proteins.
 7. Amethod for the detection of Mycobacterium bovis infection in asusceptible animal which comprises detecting in said animal an immuneresponse to MPB-70 protein of M.bovis having the amino acid sequence ofFIG. 3; said immune response being detected by contacting a serum samplefrom said animal with an antigen selected from the group consisting ofsaid MPB-70 protein and antigenic polypeptides derived therefromreactive with affinity-purified antibody to M.bovis; and detectingbinding of antibodies in said sample with said antigen to indicate thepresence of said antibodies in said sample; said MPB-70 protein orantigenic polypeptide derived therefrom being substantially free ofother M.bovis proteins.
 8. A method for the detection of Mycobacteriumbovis infection in a susceptible animal which comprises detecting insaid animal an immune response to MPB-70 protein of M.bovis having theamino acid sequence of FIG. 3; said immune response being detected bycontacting a serum sample from said animal with an antigen selected fromthe group consisting of said MPB-70 protein and antigenic polypeptidesderived therefrom reactive with affinity-purified antibody to M.bovis;and detecting binding of antibodies in said sample with said antigen bycompetition with binding of labelled antibody to said MPB-70 protein toindicate the presence of said antibodies in said sample; said MPB-70protein or antigenic polypeptide derived therefrom being substantiallyfree of other M.bovis proteins.
 9. MPB-70 protein of M.bovis having theamino acid sequence of FIG. 3, or an antigenic polypeptide derivedtherefrom reactive with affinity-purified antibody to M.bovis, saidMPB-70 protein or antigenic polypeptide derived therefrom beingsubstantially free of other M.bovis proteins.
 10. A method for thepreparation of MPB-70 protein of claim 9, which comprises purificationof an M.bovis culture filtrate by chromatofocussing.
 11. A methodaccording to claim 10, wherein said chromatofocusing step is followed byfurther purification by gel filtration.